Microtubules are one of the several cytoplasmic filaments which form the cytoskeletal framework of cells. Microtubules are also part o such specialized structures as the mitotic spindle and the axoneme of cilia and flagella. The self-assembly of microtubules and their participation in such activities as secretion, chromosome movement, saltatory movements and cell migration appear to be controlled by calcium ions and a class of proteins known as microtubule-associated proteins (MAPs). Maps may determine the site, rate and extent of polymerization of microtubules as well as the interaction of microtubules with microfilaments, intermediate filaments, the cell membrane, vesicles, cytoplasmic particles and the microtrabecular latticework in the cytoplasm. One of the most interesting problems in cell biology today is the mechanism of nucleation of microtubules at organizing centers and the control that the centers exert on the structure of microtubules. In this project, reconstituted and native microtubules will be used to study questions pertaining to the control of the protofilament number of microtubules by organizing centers, the role of MAPS as cross-bridges between microtubules and the role of calcium in the regulation of assembly. These questions will be investigated by using: (a) biochemical methods to purify and characterize microtubule proteins; (b) in vitro assembly methods to reconstitute microtubules; (c) detergent extraction procedures to isolate native spindles from eggs and tissue culture cells; and (d) EM, optical diffraction and image reconstruction techniques to resolve and enhance structural details of reassembled microtubules.